Indolopyrrolocarbazole derivatives and antitumor agents

ABSTRACT

A compound represented by the formula or a pharmaceutically acceptable salt thereof                    
     wherein R represents an unsubstituted pyridyl, furyl or thienyl group, or a pyridyl, furyl or thienyl group each of which has one or more substituents selected from the group consisting of a hydroxyl group, a lower alkoxy group, a hydroxy lower alkyl group and a hydroxy lower alkenyl group except that when the pyridyl, furyl or thienyl group has a lower alkoxy group as a substituent, each of which simultaneously has another substituent selected from the group consisting of a hydroxyl group, a lower alkoxy group, a hydroxy lower alkyl group and a hydroxy lower alkenyl group, m represents an integer of 1 to 3, and G represents a β-D-glucopyranosyl group, and the positions of substitution of the hydroxyl groups on the indolopyrrolocarbazole ring are the 1- and 11-positions, or the 2- and 10-positions, and an antitumore agent containing it as an effective ingredient. 
     The compounds have a better antitumor action than known compounds having a similar structure.

TECHNICAL FIELD

This invention is useful in the pharmaceutical field, and, moredetailedly, relates to novel indolopyrrolocarbazole derivativesinhibiting growth of tumor cells and exerting antitumor effect, andtheir use as antitumor agents.

BACKGROUND ART

Many compounds are already put to practical use as pharmaceuticals inthe field of tumor chemotherapy. However, their effects are not alwayssufficient against various kinds of tumors, and the problem ofresistance of tumor cells to these drugs also makes the methods ofclinical use complicate (see, Proceedings of the 47th Annual Meeting ofJapan Cancer Society, pages 12-15, 1988).

Under the circumstances, development of novel anticancer substances isalways desired in the field of cancer treatment. Particularly, there isneed for substances overcoming resistance to existing carcinostaticsubstances and showing effectiveness against cancers on which existingcarcinostatic substances cannot exert sufficient effect.

Under these present circumstances, the present inventors have widelyscreened metabolites of microorganisms, and as a result, they found anovel compound BE-13793C(12,13-dihydro-1,11-dihydroxy-5H-indolo[2.3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione)having antitumor activity (see, EP-A2-0388956).

Thereafter, they have tried to create compounds having further excellentantitumor activity by chemically modifying BE-13793C, and disclosed suchcompounds in prior patent applications and patents (EP-A1-0528030, U.S.Pat. Nos. 5,591,842, 5,668,271 and 5,804,564, WO95/30682 andWO96/04293).

U.S. Pat. No. 5,668,271 discloses a wide range of compounds havingantitumor activity, including compounds wherein R₁ (or R₂) is 1) a C₁-C₆alkyl group substituted by a C₆-C₁₂ aromatic hydrocarbon group; or 2) a5- or 6-membered nonaromatic or aromatic heterocyclic group. Thecompounds of the present invention have a —(CH₂)m-R group (wherein Rrepresents a substituted or unsubstituted group selected from pyridyl,furyl or thienyl) bound to the exocyclic nitrogen instead of R₁ (or R₂).Furthermore, as disclosed in the instance application, the compounds ofthe invention have much better antitumor effects than the analogouscompounds generically and specifically disclosed in U.S. Pat. No.5,668,271.

Although, in the structural formula shown in Example 35 of the aboveU.S. Pat. No. 5,668,271, a group corresponding to —(CH₂)_(m)—R in thecompounds of the present invention is shown as a 2-pyridylmethyl group,this group should be read a 2-pyridylcarbonyl group. This is apparentfrom that α-picolinohydrazide is used as a reactant.

Compounds disclosed in the above-mentioned other prior applications andpatents are further remoter in structure from the compounds of thepresent invention. Namely, EP-A1-0528030 solely discloses compoundswherein a group correspoding to —NH—(CH₂)_(m)—R in the compounds of thepresent invention is H. U.S. Pat. No. 5,591,842 discloses compoundswherein groups corresponding to —(CH₂)_(m)—R and H bound to N in thecompounds of the present invention are R¹ and R² and the R¹ and R²include comprehensive groups, but as compounds closest to the compoundsof the present invention, only compounds wherein R¹ and R² are C₁-C₆alkyl groups substituted with a C₆-C₁₂ aromatic hydrcarbonic group, orfuryl, thienyl or pyridyl groups (one of R¹ and R² can be H) aredisclosed. U.S. Pat. No. 5,804,564 and WO95/30682 solely disclosecompounds wherein a group corresponding to —(CH₂)_(m)—R in the compoundsof the present invention is a bis(hydroxymethyl)methyl group. WO96/04293discloses compounds wherein a group corresponding to —NH(CH₂)_(m)—R inthe compounds of the present invention is R¹ and this R¹ includescomprehensive groups, but compounds close to the compounds of thepresent invention are not found and, further, a group corresponding to Gin the compounds of the present invention is a disaccharide group.

Some of the compounds of the present invention, namely compounds whereinR is a pyridyl, furyl or thienyl group each having a substituent aredisclosed in JP-A-10-245390 (published on Sep. 14, 1998), the inventorsof which are the applicants of the present application. The JP-Aapplication has not yet been examined and applications to othercountries corresponding thereto have not been made except the presentapplication.

From the above, in relation with the above-mentioned background art, theinvention of the present application are believed not to be consideredat least as an object of rejection under 35 U.S.C. §102 (B) and (D).

DISCLOSURE OF INVENTION

It is a problem to be solved in the present invention to createcompounds having further excellent antitumor activity by chemicallymodifying indolopyrrolocarbazole antitumor substances disclosed in theprior patent applications.

For solving the above problem, the present inventors have synthesized awide range of indolopyrrolocarbazole derivatives and examined theirantitumor or activity, and found that compounds represented by thelater-described formula [I] show further excellent antitumor activitythan the indolopyrrolocarbazole compounds disclosed in the priorapplications, and completed the present invention.

Namely, the invention relates to a compound represented by the formulaor a pharmaceutically acceptable salt thereof

wherein R represents an unsubstituted pyridyl, furyl or thienyl group,or a pyridyl, furyl or thienyl group each of which has one or moresubstituents selected from the group consisting of a hydroxyl group, alower alkoxy group, a hydroxy lower alkyl group and a hydroxy loweralkenyl group except that when the pyridyl, furyl or thienyl group has alower alkoxy group as a substituent, each of which simultaneously hasanother substituent selected from the group consisting of a hydroxylgroup, a lower alkoxy group, a hydroxy lower alkyl group and a hydroxylower alkenyl group, m represents an integer of 1 to 3, and G representsa β-D-glucopyranosyl group, and the positions of substitution of thehydroxyl groups on the indolopyrrolocarbazole ring are the 1- and11-positions, or the 2- and 10-positions, and a use thereof as anantitumor agent.

BEST MODE FOR CARRYING OUT THE INVENTION

In the definition of the substituents in the compounds of the inventionrepresented by the formula [I], lower alkoxy groups representstraight-chain or branched alkoxy groups having 1 to 6, preferably 1 to4, further preferably 1 or 2, most preferably 1 carbon atoms, and therecan for example be mentioned a methoxy group, an ethoxy group, a propoxygroup, n-butoxy group, s-butoxy group, n-hexyl group, etc.

Hydroxy lower alkyl groups represent straight-chain or branchedhydroxyalkyl groups having 1 to 6, preferably 1 to 4, further preferably1 or 2, most preferably 1 carbon atoms, and there can for example bementioned a hydroxymethyl group, a hydroxyethyl group, a hydroxypropylgroup, a hydroxybutyl group, a hydroxyhexyl group, etc.

Hydroxy lower alkenyl groups represent straight-chain or branchedhydroxyalkenyl groups having 2 to 6, preferably 3 or 4, furtherpreferably 3 carbon atoms, and there can for example be mentioned a3-hydroxy-1-propenyl group, etc. m represents an integer of 1 to 3,preferably 1.

The positions of substitution of the hydroxyl groups on theindolopyrrolocarbazole ring may be the 1- and 11-positions, or the 2-and 10-positions, but the 2- and 10-positions are preferred.

Description is made below on processes for preparing the compounds ofthe invention.

An indolopyrrolocarbazole derivative of the invention can be prepared byreacting a compound represented by the formula

wherein, A represents NH or H, and G is as defined above, the compoundbeing a known compound disclosed in EP-A1-0528030, EP-A1-0545195,WO95/30682 and WO96/04293, with a compound represented by the formula

H₂N—NH(CH₂)_(m)R  [III]

wherein, R and m are as defined above, or by condensing a compoundrepresented by the formula

wherein, G is as defined above, with a compound represented by theformula

 R¹(CH₂)_(m)CHO  [V]

wherein, R¹ has the same meaning as R or means R wherein hydroxyl groupsare protected, and m is as defined above, then carrying out reduction,and, if necessary, removing the protective groups, or by reacting acompound of the formula [IV] with a compound represented by the formula

R¹(CH₂)_(m)L  [VI]

wherein, L means a leaving group, and R¹ and m are as defined above,and, if necessary, removing the protective groups.

The reaction between the compound represented by the formula [II] andthe compound represented by the formula [III] is reaction between animide or an acid anhydride and a hydrazine derivative, well-known in thechemical field. This reaction can be carried out using a solvent usuallyhaving no bad influenece on the reaction, such as, for example,tetrahydrofuran or N,N-dimethylformamide. The use amount of the compound[III] is usually a little excess to 5 molar equivalents based on thecompound [II], but a largely excess use of the former is possible.

The reaction temperature is usually in the range of −50° C. to theboiling point of the solvent, but, if necessary, temperature higher thanor lower than the temperature can be used. The reaction time is usuallyin the range of 30 minutes to 2 days, but time longer than or shorterthan the time can be used.

The reaction of preparing a compound [I] by condensing a compoundrepresented by the formula [IV] with a compound represented by theformula [V] and then carrying out reduction can be carried out in thesame reaction system, but in some occasion, it is also possible to, onceisolate the Schiff base as an intermediate product. Namely usually, thereaction can be carried out by mixing the compound [IV] with thecompound [V] in a suitable solvent and then adding a reducing agent. Thereaction is preferably carried out in the presence of an acid such asacetic acid or hydrochloric acid. As usable solvents, there can, forexample, be mentioned alcoholic solvents such as methanol and ethanol,aprotic polar solvents such as N,N-dimethylformamide, etc. The reductionof the Schiff base can be carried out using a metal hydride complex suchas sodium cyanoborohydride, or the like, and also by catalyticreduction.

The reaction between a compound [IV] and a compound [VI] is alkylationreaction of an amine, and can be carried out by a known method, forexample by reaction with an alkyl halide, alkyl mesylate or alkyltosylate or the like.

The products of the above reactions can be purified by methods known inthe field of organic chemistry, for example by precipitation methods,solvent extraction methods, recrystallization, chromatography, etc.

Further in the invention, pharmaceutically acceptable salts of compoundsobtained by the above processes are included. As such salts, there canbe mentioned salts with an alkali metal such as for example potassium orsodium, salts with an alkaline earth metal such as for example calcium,salts with a basic organic compound such as for example ethylamine orarginine, salts with an inorganic acid such as hydrochloric acid orsulfuiric acid, and salts with an organic acid such as acetic acid,citric acid or maleic acid.

The compounds of the invention represented by the formula [I] showexcellent antitumor action.

Growth Inhibition Activity on Mouse Leukemia Cells

100 μl of a medium for cell culture (RPMI-1640 medium containing 10%fetal bovine serum) containing 3×10³ mouse leukemia cells (P388) was putin a 96-well microplate, and culture was carried out at 37° C. for 24hours under 5% CO₂. 10 μl of a medium containing a test compound wasadded, and culture was continued at 37° C. for further 24 hours under 5%CO₂. 10 μl of 0.5% Thiazoyl Blue was added to the cultured medium, andincubation was made at 37° C. for 2 hours under 5% CO₂ to carry outenzymatic reaction. 20% sodium dodecyl sulfate (SDS) was added todiscontinue the reaction, and incubation was carried out at 37° C. forfurther 4 hours to dissolve the resulting dye, and absorbance at 550 nmwas measured and compared with the control group. The results are shownin Table 1.

Growth Inhibition Activity on Human Gastric Cancer Cells, Human ColonCancer Cells and Human Lung Cancer Cells

100 μl of a medium for cell culture (RPMI-1640 medium containing 10%fetal bovine serum) containing 1×10⁴ human gastric cancer cells(MKN-45), human colon cancer cells (DLD-1) or human lung cancer cells(PC-13) was put in a 96-well microplate, and culture was carried out at37° C. for 24 hours under 5% CO₂. 100 μl of a medium containing a testcompound was added, and culture was continued at 37° C. for further 72hours under 5% CO₂. After completion of the culture, 50 μl of 50%trichloroacetic acid was added to the cultured medium, and the mixtureswere held at 4° C. for 60 minutes to fix the cells. The cultured mediumcontaining trichloroacetic acid was removed, and the wells were washedwith tap water and dried. 50 μl of 0.4% Sulforhodamine B solution wasadded, and the mixtures were held at room temperature for 30 minutes todye the cells. The dyeing solutions were removed, and the remainingdyeing solutions were washed away with 1% acetic acid. After completeremoval of the acetic acid solution and drying, 200 μl of 10 nM Trissolution was added and the mixtures were mixed well at room temperaturefor 1 hour or more to extract the dye. Absorbance of each well at 560 nmwas measured, and the inhibition proportion in comparison with the groupof no addition of any drug was calculated. The results are shown inTable 1.

TABLE 1 Growth inhibition activity on some tumor cells 50% inhibitionconcentration (IC₅₀, nM) Test compound P-388 MKN-45 DLD-1 PC-13 Example3 0.28 0.35 0.91 0.22 15 0.50 2.10 4.3 0.62 16 0.40 1.30 4.0 0.56 180.43 0.66 4.6 0.30 19 0.19 0.92 1.3 0.50 20 0.87 0.92 1.0 0.67 22 0.631.80 1.0 0.58 23 0.48 1.50 8.9 2.3 24 0.48 0.71 3.3 0.27 Control 190 398200 710 compound 1 Control 6.5 12.0 76 48.0 compound 2

Control compound 1: a compound of the formula [I] of the presentinvention wherein —AAAAAAA(CH₂)_(m)—R is a 2-pyridyl group and thepositions of substitution of the hydroxyl groups on theindolopyrrolocarbazole ring are the 1- and 11-positions. The controlcompound 1 is disclosed in Example 15 of U.S. Pat. No. 5,668,271.

Control compound 2: a compound of the formula [I] of the presentinvention wherein —(CH₂)_(m)—R is a 3-hydroxybenzyl group and thepositions of substitution of the hydroxyl groups on theindolopyrrolocarbazole ring are the 1- and 11-positions. The controlcompound 1 is disclosed in Example 23 of U.S. Pat. No. 5,668,271.

Antitumor Effect on Human Gastric Cancer (MKN-45), Human Breast Cancer(MX-1) and Human Colon Cancer (LS180)

A MKN-45, MX-1 or LS180 solid tumor previously subcutaneously implantedin a nude mouse and grown was thinly cut, and its cubes with each side 3mm were subcutaneously implanted in test mice. After the implantation,starting from the time when the tumor grew into 0.3 cm³ or more, variousdosages of a test compound were injected once a day for 5 consecutivedays into the tail veins of the mice. The same injections were then madefor further 5 days after 2 days of interval (treatment schedule: 5/w×2)or once every 3-4 days, four times in total (treatment schedule: 2/w×2).28 after the start of the treatment, the length (L) and the breadth (W)of each of the tumors were measured, and its volume (V) was calculated(V=½×L×W²). A tumor growth inhibition proportion was calculated based onthe volume, and a total dose to inhibit tumor growth by 75% (GID₇₅,mg/m² body surface area) was then determined. The results are shown inTable 2.

TABLE 2 Antitumor action on mice carrying various tumors GID₇₅(mg/m²total) Human Test Human gastric cancer Human breast cancer colic cancercompound MKN-45 MX-1 LS180 Example 3 <30 <12 48 15 <90 <6 400 16 <36 1816 19 <30 20 <30 23 19 24  12 27 114 Control 370 97 1900 compound

Control compound: a compound of the formula [I] of the present inventionwherein —(CH₂)_(m)—R is a 3-hydroxybenzyl group and the positions ofsubstitution of the hydroxyl groups on the indolopyrrolocarbazole ringare the 2- and 10-positions. This control compound is not specificallydisclosed in U.S. Pat. No. 5,668,271, but is an isomer of the compounddisclosed in Example 23 of the U.S. Patent, both compounds differing inthe positions of substitution of the hydroxyl groups on theindolopyrrolocrbazole ring.

Compounds provided by the present invention show a much better antitumoraction than the control compounds, as shown in the above pharmacologicaltest results.

As apparent from the results of the above pharmacological tests, thecompounds of the invention show an excellent antitumor action, and areuseful as antitumor agents for humans or other mammals, particularlyhumans, for prophylaxis and/or treatment of, for example, head or neckcancer, thyroid cancer, lung cancer, esophageal cancer, gastric cancer,hepatic cancer, pancreatic cancer, colon cancer, renal cancer, prostatecancer, testoid cancer, uterine cancer, ovarian cancer, breast cancer,brain cancer, etc. as solid cancers, and leukemia, lymphoma, myeloma,etc. as other cancers, preferably gastric cancer, colon cancer, lungcancer and breast cancer.

A compound of the invention can be used in the form of antitumorpharmaceutical preparations suitable for oral administration, parenteraladministration, etc., obtained by mixing the compound with solid orliquid excipients or carriers known in the field of pharmaceuticalpreparations. As forms for oral administration, there can be mentionedperoral agents such as for example tablets, capsuls, powders, granulesand liquids, and as forms for parenteral administration, there can bementioned sterilized liquid parenteral agents such as for examplesolutions and suspensions.

Solid preparations such as tablets, capsules, granules and powders canbe prepared using compounds of the invention alone, but can also beprepared further using suitable additives. As the suitable additives,there can be mentioned conventional additives, for example, sugars suchas for example lactose and glucose, starches such as for example corn,wheat and rice, fatty acids such as for example stearic acid, inorganicsalts such as for example magnesium metasilicate aluminate and anhydrouscalcium phosphate, synthetic macromolecules such as for examplepolyvinylpyrrolidone and polyalkylene glycols, fatty acid salts such asfor example calcium stearate and magnesium stearate, alcohols such asfor example stearyl alcohol and benzyl alcohol, synthetic cellulosederivatives such as for example methylcellulose, carboxymethylcellulose,ethylcellulose and hydroxypropylmethylcellulose, and further, water,gelatin, talc, vegetable oils, gum arabic, etc.

These solid preparations such as tablets, capsules, granules and powderscan contain, generally 0.1 to 100% by weight, preferably 5 to 100% byweight of an effective ingredient.

Liquid preparations can be prepared as forms of suspensions, syrups,injections, etc. using suitable additives usually used in liquidpreparations, such as water, alcohols or vegetable oils includingsoybean oil, peanut oil and sesame oil.

Particularly, as solvents suitable in parenteral administrationincluding intramuscular injection, intravenous injection andsubcutaneous injection, there can for example be mentioned distilledwater for injection, aqueous lidocaine hydrochloride solution (forintramuscular injection), physiological saline, aqueous glucosesolution, ethanol, polyethylene glycol, liquids for intravenousinjection (e.g., aqeous solutions of cetric acid and sodium citrate,etc.), electrolyte solutions (for intravenous injection by drip and forintravenous injection), etc., or their mixed solutions.

These injections may include not only those wherein previous dissolutionis made, but also those in the form of dissolving powder alone or withsuitable additives when used. These injections usually contain 0.1 to10% by weight, preferably 1 to 5% of an effective ingredient.

Liquid preparations such as suspensions and syrups for oraladministration can contain 0.5 to 10% by weight of an effectiveingredient.

It should be noted that the actually preferred dose of the compounds ofthe invention is varied depending on kinds of compounds used, kinds ofcompositions prepared, application frequency, particular sites to betreated, hosts and tumors. For example, the dose of each compound perday and per one adult is 10 to 500 mg in the case of oraladministration, and 10 to 100 mg in the case of parenteraladministration, preferably intravenous injection. The frequency ofadministration is varied depending on administration methods andsymptoms, but the administration can be made in a time or in devided 2to 5 times. Administration methods including intermittent administrationsuch as every two-days administration or every three-days administrationcan also be used.

EXAMPLES

The invention is further specifically described below according toexamples, but the invention is not limited to these examples.

Compounds having the following structural formulae, which were used asstarting compounds, are hereinafter referred to as follows.

wherein, G represents a β-D-glucopyranosyl group, which is the same inthe following examples.

Example 1

Compound represented by the structural formula

30 mg of Compound A and 30 mg of(5-t-butyldimethylsilyloxymethylthiophene)-2-carboxyaldehyde weredissolved in 6 ml of methanol, 30 ml of acetic acid was added, and themixture was stirred at 80° C. for 4 hours. The reaction mixture wascooled to room temperature, 20 mg of sodium cyanoborohydride and 200 mlof a 10% solution of hydrochloric acid in methanol were added, and themixture was stirred at room temperature for 30 minutes. The reactionmixture was concentrated to dryness under reduced pressure, and theresidue was put on a Sephadex LH-20 column for chromatography and elutedwith methanol. Fractions containing the desired compound wereconcentrated to dryness to obtain 27 mg of the compound represented bythe above formula.

Rf value: 0.37 (Kieselgel 60F₂₅₄ made by Merck Co., developing solvent;acetonitrile:tetrahydrofuran:toluene:water:acetic acid=4:2:2:0.5:0.1)

FAB-MS(m/z):660(M⁺)

¹H-NMR(300 MHz,DMSO-d₆, δ ppm):11.19(1H,s),9.79(1H,s),9.75(1H,s),8.86(1H,d,J=9.3 Hz),8.78(1H,d,J=9.0 Hz),7.17(1H,d,J=2.1Hz),6.97(1H,d,J=2.1 Hz),6.89(1H,d,J=3.6 Hz),6.82(1H,dd,J=2.1,9.3Hz),6.79(1H,dd,J=2.1,9.0 Hz),6.73(1H,d,J=3.3 Hz),6.10(1H,t,J=4.5Hz),5.97(1H,d,J=8.1 Hz),5.86(1H,t,J=3.3 Hz),5.35(1H,t,J=6.0Hz),5.32(1H,d,J=4.8 Hz),5.12(1H,d,J=4.8 Hz),4.92(1H,d,J=5.4Hz),4.52(2H,d,J=5.7 Hz),4.40(2H,d,J=4.2Hz),4.02(1H,m),3.91(2H,m),3.78(1H,m),3.50(2H,m)

Example 2

Compound represented by the structural formula

43 mg of Compound A and 100 mg of(3-t-butyldimethylsilyloxymethyl)-3-pryidinecarbaldehyde were suspendedin 10 ml of methanol, 18 ml of acetic acid was added, and the mixturewas stirred overnight at 80° C. The reaction mixture was concentrated,put on a Sephadex LH-20 column for chromatography and eluted withmethanol. Fractions containing the desired compound were concentrated todryness, and the residue was dissolved in 5 ml of a mixed solvent ofmethanol/tetrahydrofuran (1:1). 5% palladium-carbon was added and themixture was stirred at room temperature for 3.5 hours under a hydrogenstream. The reaction mixture was filtered using Celite and the residuewas dissolved in 5 ml of tetrahydrofuran. An excess amount oftetrabutylammonium fluoride (1.0 M tetrahydrofuran solution) was addedand the mixture was stirred at room temperature for 30 minutes. Thereaction mixture was concentrated, put on a Sephadex LH-20 column forchromatography, and eluted with methanol. Fractions containing thedesired compound were concentrated to dryness to obtain 4.3 mg of thecompound represented by the above formula. Rf value: 0.1 (Kieselgel60F₂₅₄ made by Merck Co., developing solvent;acetonitrile:tetrahydrofuran:toluene:water:acetic acid=4:2:2:0.5:0.1)

FAB-MS(m/z):656(M+H)⁺

¹H-NMR(300 MHz,DMSO-d₆, δppm):11.18(1H,s),9.75(1H,s),9.73(1H,s),8.84(1H,d,J=8.4Hz),8.77(1H,d,J=8.9 Hz),8.42(1H,d,J=4.9 Hz),8.39(1H,s),7.48(1H,d,J=4.9Hz),7.17(1H,d,J=1.1 Hz),6.97(1H,s),6.78-6.83(2H,m),609(1H,t,J=4.7Hz),5.97(1H,d,J=6.6 Hz),5.84(1H, t,J=3.8 Hz),5.39(1H,t,J=5.8Hz),5.30(1H,d,J=4.8 Hz),5.09(1H,d,J=4.2 Hz),4.95(2H,d,J=5.3Hz),4.90(1H,d,J=3.3 Hz),4.27(2H,d,J=4.2Hz),3.75-4.03(4H,m),3.47-3.52(2H,m)

Example 3

Compound represented by the structural formula

98 mg of Compound A and 92.1 mg of4-(3-t-butoxymethyl)pyridinecarbaldehyde were dissolved in 5 ml ofmethanol, 18 ml of acetic acid was added, and the mixture was stirredovernight at 80° C. The reaction mixture was concentrated, and theresulting crystals were washed with chloroform and dissolved in a mixedsolvent of methanol/tetrahydrofuran (1:1). 5% palladium-carbon was addedand the mixture was stirred for 3 hours under a hydrogen stream. Thereaction mixture was filtered using Celite and the filtrate wasconcentrated. The residue was dissolved in tetrahydrofuran,tetrabutylammonium fluoride was added, and the mixture was stirred atroom temperature for 30 minutes. Water was added, the mixture wasextracted with methyl ethyl ketone, and the organic layer was washedwith an aqueous saturated sodium chloride solution and concentrated. Theconcentrate was put on a Sephadex LH-20 column for chromatography andeluted with methanol. Fractions containing the desired compound wereconcentrated to dryness to obtain 13.5 mg of the compound represented bythe above formula.

Rf value: 0.10 (Kieselgel 60F₂₅₄ made by Merck Co., developing solvent;chloroform:methanol:tetrahydrofuran=2:2:1)

FAB-MS(m/z): 656(M+H)⁺

¹H-NMR(300 MHz,DMSO-d₆, δppm):11.19(1H,s),9.78(1H,s),9.75(1H,s)8.85(1H,d,J=9.1Hz),8.77(1H,d,J=9.1 Hz),8.51(1H,s),8.11(1H,d,J=5.1 Hz),7.59(1H,d,J=4.6Hz),7.17(1H,d,J=2.1 Hz),6.97(1H,d,J=1.8Hz),6.79-6.85(2H,m),6.25(1H,t,J=5.0 Hz),5.98(1H, d,J=8.3Hz),5.86(1H,d,J=4.5 Hz),5.32(1H,d,J=4.5 Hz),5.23(1H,t,J=5.6Hz),5.11(1H,d,J=4.4 Hz),4.91(1H,d,J=4.9 Hz),4.74(2H,d,J=5.2Hz),4.35(2H,d,J=7.8 Hz),3.73-4.05(4H,m),3.43-3.52(2H,m)

Example 4

Compound represented by the structural formula

50 mg of Compound A and 100 mg of(4,5-t-butyldimethylsilyloxymethyl)thiophene-2-carboxyaldehyde weresuspended in 10 ml of anhydrous methanol, 100 ml of acetic acid wasadded, and the mixture was stirred at 80° C. for 2 hours. The reactionmixture was put on a Sephadex LH-20 column for chromatography and elutedwith methanol. Fractions containing the desired compound wereconcentrated to dryness to obtain 40 mg of an intermediate compound.This was suspended in 3 ml of methanol, 12 mg of sodium cyanoborohydrideand and 300 ml of a 10% solution of hydrochloric acid in methanol wereadded, and the mixture was stirred at room temperature for 1 hour. Thereaction mixture was put on a Sephadex LH-20 column for chromatographyand eluted with methanol. Fractions containing the desired compound wereconcentrated to dryness, and the residue was purified by preparativethin layer chromatography (Kieselgel 60F₂₅₄ made by Merck Co.,developing solvent; acetonitrile:tetrahydrofuran:toluene:water:aceticacid=4:2:2:0.5:0.1) to obtain 26 mg of the compound represented by theabove formula.

Rf value: 0.28 (Kieselgel 60F₂₅₄ made by Merck Co., developing solvent;acetonitrile:tetrahydrofuran:toluene:water:acetic acid=4:2:2:0.5:0.1)

FAB-MS(m/z):690(M⁺)

¹H-NMR(300 MHz,DMSO-d₆, δppm):11.17(1H,s),9.50-10.15(2H,br),8.86(1H,d,J=8.4 Hz),8.78(1H,d,J=8.7Hz),7.16(1H,d,J=1.8 Hz),6.97(1H,d,J=2.1Hz),6.92(1H,s),6.82(1H,dd,J=1.8,8.7 Hz),6.79(1H,dd,J=2.1,8.4Hz),6.04(1H,t,J=5.1 Hz),6.04(1H,t.J=5.1 Hz),5.96(1H,d,J=8.1Hz),5.88(1H,br),5.35(1H,br),5.28(1H,br),5.15(1H,br),4.93(2H,br),4.53(2H,br),4.73(2H,d,J=4.5Hz),4.30(1H,s),4.00(1H,m),3.91(2H,m),3.77(1H,m),3.52(2H,m)

Example 5

Compound represented by the structural formula

30 mg of Compound A and 50 mg of 2-(3-t-butyldimethylsilyloxymethyl)pyridinecarbaldehyde were suspended in 6 ml of methanol, 30 ml of aceticacid was added, and the mixture was stirred at 80° C. for 2 hours. Thereaction mixture was put on a Sephadex LH-20 column for chromatographyand eluted with methanol. Fractions containing the desired compound wereconcentrated to obtain 40 mg of an intermediate compound. This wasdissolved in a mixed solvent of tetrahydrofuran/methanol (2:1), 15 mg ofsodium cyanoborohydride and 3 ml of a solution of hydrochloric acid inmethanol were added, and the mixture was stirred at room temperature for3 hour. The reaction mixture was concentrated, put on a Sephadex LH-20column for chromatography and eluted with methanol. Fractions containingthe desired compound were concentrated to dryness to obtain 27 mg of thecompound represented by the above formula.

Rf value: 0.12 (Kieselgel 60F₂₅₄ made by Merck Co., developing solvent;acetonitrile:tetrahydrofuran:toluene:water:acetic acid=4:2:2:0.5:0.1)

FAB-MS(m/z):656(M+H)³⁰

¹H-NMR(300 MHz,DMSO-d₆, δ ppm):11.18(1H,br),9,77(2H,br,)8.82(1H,d, J=9.0Hz),8.784(1H,d,J=9.0 Hz),8.27(1H,dd,J=1.8,5.1 Hz),7.83(1H,d,J=8.1Hz),7.28(1H,dd,J=8.1,5.1 Hz),7.17(1H,d,J=1.8 Hz),6.98(1H,d,J=1.8Hz),6.82(1H,dd,J=1.8,9.1 Hz),6.79(1H,dd,J=1.8,9.0 Hz),6.15(1H,t,J=5.1Hz),5.97(1H,d,J=8.1 Hz),5.86(1H,t,J=4.2 Hz),5.33(1H,d,J=5.4Hz),5.31(1H,d,J=5.4 Hz),5.12(1H,d,J=4.8 Hz),4.93(1H,d,J=4.5Hz),4.87(2H,d,J=6.0Hz),4.36(2H,d,J=5.1Hz),4.03(1H,m),3.91(2H,s),3.79(1H,m),3.51(2H,m)

Example 6

Compound represented by the structural formula

30 mg of Compound C and 65 mg of(6-hydroxymethyl-3-pyridylmethyl)hydrazine hydrochloride were dissolvedin 5 ml of N,N-dimethylformamide, 0.5 ml of triethylamine was added, andthe mixture was stirred at 80° C. for 3 hours. 33 mg of(6-hydroxymethyl-3-pyridylmethyl)hydrazine hydrochloride was added andthe mixture was stirred at 80° C. for 2 hours. The reaction mixture wasconcentrated to dryness, and the residue was put on a Sephadex LH-20column for chromatography and eluted with methanol. Fractions containingthe desired compound were concentrated, and the residue was again put ona Sephadex LH-20 column for chromatography and eluted with ethanol.Fractions containing the desired compound were concentrated to drynessto obtain 7.2 mg of the compound represented by the above formula.

FAB-MS(m/z):656(M+H)⁺

¹H-NMR(300 MHz,DMSO-d₆, δ ppm):11.12(1H,br),8.81(1H,d,J=8.7Hz),8.73(1H,d,J=8.7 Hz),8.51(1H,s),7.90(1H,d,J=7.9 Hz),7.39(1H,d,J=7.9Hz),7.11(1H,s),6.93(1H,s),6.77(2H,t,J=8.7 Hz),6.21(1H,t,J=3.8Hz),5.92(1H,d,J=7.9 Hz),4.85-5.50(5H,br),4.48(2H,s),4.27(2H,d,J=3.8Hz),3.70-4.05(4H,m),3.45-3.52(2H,m)

Example 7

Compound represented by the structural formula

12.5 mg of Compound C and 42 mg of(5-hydroxymethyl-3-pyridylmethyl)hydrazine hydrochloride were dissolvedin 1 ml of N,N-dimethylformamide, 0.1 ml of triethylamine was added, andthe mixture was stirred at 80° C. for 2.5 hours. 0.1 ml of triethylaminewas added and the mixture was stirred overnight at 50° C. The reactionmixture was concentrated to dryness, and the residue was put on aSephadex LH-20 column for chromatography and eluted with methanol.Fractions containing the desired compound were concentrated, and theresidue was again put on a Sephadex LH-20 column for chromatography andeluted with ethanol. Fractions containing the desired compound wereconcentrated to dryness to obtain 2.4 mg of the compound represented bythe above formula.

Rf value: 0.18 (Kieselgel 60F₂₅₄ made by Merck Co., developing solvent;acetonitrile:tetrahydrofuran:toluene:water:acetic acid=4:2:2:0.5:0.1)

FAB-MS(m/z):656(M+H)⁺

¹H-NMR(300 MHz,DMSO-d₆, δppm):11.18(1H,br),9.60-10.02(2H,br),8.84(1H,d,J=8.5 Hz),8.76(1H,d,J=8.6Hz),8.55(1H,s),8.37(1H,s),7.84(1H,s),7.16(1H,s),7.00(1H,s),6.75-6.85(2H,m),6.21(1H,t,J=4.7Hz),5.95(1H,d,J=7.8Hz),5.88-5.95(1H,br),5.40-5.48(1H,br),5.26-5.35(1H,br),5.15-5.25(1H,br),4.90-4.93(1H,br),4.56(2H,d,J=4.7Hz),4.50(2H, s),3.72-4.05(4H,m),3.45-3.55(2H,m)

Example 8

Compound represented by the structural formula

30 mg of Compound D and 65 mg of(6-hydroxymethyl-3-pyridylmethyl)hydrazine hydrochloride were dissolvedin 5 ml of N,N-dimethylformamide, 0.5 ml of triethylamine was added, andthe mixture was stirred at 80° C. for 1.5 hours. The reaction mixturewas dried and concentrated, and the residue was put on a Sephadex LH-20column for chromatography and eluted with methanol. Fractions containingthe desired compound were concentrated to dryness to obtain 29 mg of thecompound represented by the above formula.

FAB-MS(m/z):656(M+H)⁺

¹H-NMR(300 MHz,DMSO-d₆, δ ppm):10.89(1H,br),10.36(1H,br),9.97(1H,br),8.67(1H,d,J=7.9 Hz),8.52(1H,d,J=2.2 Hz),8.50(1H,d,J=7.9Hz),7.93(1H,dd,J=2.2,8.1 Hz),7.41(1H,d,J=8.1 Hz),7.18(2H,t,J=7.9Hz),7.02(1H,d,J=7.9 Hz),7.00(2H,t,J=7.9 Hz),6.29(1H,t,J=4.5Hz),5.42(1H,d,J=5.6 Hz),5.33(1H,d,J=6.1 Hz),5.32(1H,t,J=6.0Hz),5.21(1H,d,J=5.3 Hz),4.82-4.91(1H,br),4.48(2H,d,J=6.0Hz),4.29(2H,d,J=4.5 Hz),3.91-4.12(2H,m),3.52-3.79(3H,m),3.30-3.40(1H,m)

Example 9

Compound represented by the structural formula

30 mg of Compound B and 152 mg of4-(t-butyldimethylsilyloxyethyl)thiophene-2-carbaldehyde were suspendedin 6 ml of anhydrous methanol, 30 ml of acetic acid was added, and themixture was stirred at 80° C. for 2 hours. The reaction mixture was puton a Sephadex LH-20 column for chromatography and eluted with methanol.Fractions containing the desired compound were concentrated to drynessto obtain 31 mg of an intermediate compound. This was suspended in 3 mlof methanol, 30 mg of sodium cyanoborohydride and 300 ml of a 10%solution of hydrochloric acid in methanol were added, and the mixturewas stirred at room temperature for 1 hour. The reaction mixture wasdiluted with ethyl acetate, washed with water and an aqueous saturatedsodium chloride solution, dried and concentrated. The residue was put ona Sephadex LH-20 column for chromatography and eluted with methanol.Fractions containing the desired compound were concentrated to drynessto obtain 5 mg of the compound represented by the above formula.

Rf value: 0.43 (made by Merck Co., toluene:water:aceticacid=4:2:2:0.5:0.1)

FAB-MS(m/z):675(M)⁺

¹H-NMR(300 MHz,DMSO-d₆, δ ppm):11.19(1H,s),9.80(2H,br),8.86(1H,d,J=9.0Hz),8.78(1 H,d,J=8.7 Hz),7.17(1H,d,J=1.8 Hz),7.03(1H,s),6.98(1H,d,J=2.1Hz),6.95(1H,s),6.80(2H,dt,J=2.1,8.7 Hz),6.08(1H,t,J=5.1Hz),5.96(1H,d,J=7.8Hz),5.89(1H,br),5.36(1H,br),5.13(1H,br),4.93(1H,br),4.57(1H,br),4.38(2H,d,J=4.2Hz),4.05(2H,m),3.92(2H,s),3.77(1H,m),3.50(5H,m)

Example 10

Compound represented by the structural formula

17 mg of Compound D and 12 mg of4-(2-hydroxymethyl-4-pyridylmethyl)hydrazine trifluoroacetate weredissolved in 2 ml of N,N-dimethylformamide, 0.1 ml of triethylamine wasadded, and the mixture was stirred at 75° C. for 2 hours. Water andethyl acetate were added to the reaction mixture, and the mixture wasextracted three times with water. Sodium chloride was added to theaqueous layer and the mixture was extracted three times with methylethyl ketone. The organic layer was dried and concentrated, and theresidue was put on a Sephadex LH-20 column for chromatography and elutedwith methanol. Fractions containing the desired compound wereconcentrated to dryness to obtain 8 mg of the compound represented bythe above formula.

FAB-MS(m/z):656(M+H)⁺

¹H-NMR(300 MHz,DMSO-d₆, δ ppm):10.90(1H,br),8.66(1H,d,J=7.6Hz),8.50(1H,d,J=7.6 Hz),8.41(1H,d,J=5.0 Hz),7.57(1H,s),7.48(1H,d,J=5.0Hz),7.17(2H,t,J=7.6 Hz),7.07(1H,d,J=7.6 Hz),7.00(1H,d,J=7.6Hz),6.98(1H,t,J=7.6 Hz),6.32(1H,t,J=4.8 Hz),5.36(1H,t,J=3.7Hz),5.10-5.50(4H,br),4.51(2H,d,J=3.7 Hz),4.34(2H,d,J=4.8Hz),3.91-4.12(2H,m),3.51-3.80(3H,m)

Example 11

Compound represented by the structural formula

17 mg of Compound C and 12 mg of(2-hydroxymethyl-4-pyridylmethyl)hydrazine trifluoroacetate weredissolved in 1 ml of N,N-dimethylformamide, 0.1 ml of triethylamine wasadded, and the mixture was stirred at 80° C. for 3.5 hours. Water andethyl acetate were added to the reaction mixture, and the mixture wasseparated into two layers. Sodium chloride was added to the aqueouslayer and the mixture was extracted with methyl ethyl ketone. Theorganic layer was dried and concentrated, and the residue was put on aSephadex LH-20 column for chromatography and eluted with methanol.Fractions containing the desired compound were concentrated to drynessto obtain 4 mg of the compound represented by the above formula.

FAB-MS(m/z):656(M+H⁺)

¹H-NMR(300 MHz,DMSO-d₆, δppm):11.17(1H,br),9.55-10.05(2H,br),8.85(1H,d,J=8.4 Hz),8.77(1H,d,J=8.2Hz),8.41(1H,d,J=5.1 Hz),7.56(1H,s),7.47(1H,d,J=5.1Hz),7.15(1H,s),6.96(1H,s),6.72-6.85(2H,m),6.26(1H,t,J=4.9Hz),5.94(1H,d,J=8.6Hz),5.80-5.99(1H,br),5.30-5.42(2H,br),5.10-5.20(1H,br),4.85-4.95(1H,br),4.51(2H,d,J=1.8Hz),4.32(2H,d,J=4.5Hz),3.89-4.04(1H,m),3.90(2H,m),3.74-3.78(1H,m),3.50(2H,m)

Example 12

Compound represented by the structural formula

14 mg of Compound A and 14.7 mg of5-t-butyldimethylsilyloxymethylpyridine-2-carbaldehyde were suspended in2 ml of anhydrous methanol, 8 ml of acetic acid was added, and themixture was stirred overnight at 80° C. The reaction mixture was put ona Sephadex LH-20 column for chromatography and eluted with methanol.Fractions containing the desired compound were concentrated to drynessto obtain 15.3 mg of an intermediate compound. 75 mg of sodiumcyanoborohydride was suspended in 1 ml of tetrahydrofuran, and 0.55 mlof zinc chloride (1.0 M diethyl ether solution) was added dropwise. Asuspension of 15.3 mg of the intermediate compound in 3 ml oftetrahydrofuran was added, and the mixture was stirred at roomtemperature for 2.5 hours. An aqueous saturated sodium bicarbonatesolution was added to the reaction mixture, and the mixture wasextracted with methyl ethyl ketone. The organic layer was dried andconcentrated, the residue was dissolved in 3 ml of tetrahydrofuran, andan excess amount of tetrabutylammonium fluoride (1 M tetrahydrofuransolution) was added dropwise at 0° C. The mixture was stirred at roomtemperature for 30 minutes, water was added, and the mixture wasextracted with methyl ethyl ketone. The organic layer was dried andconcentrated, and the residue was put on a Sephadex LH-20 column forchromatography and eluted with methanol. Fractions containing thedesired compound were concentrated to dryness to obtain 4.5 mg of thecompound represented by the above formula.

Rf value: 0.1 (Kieselgel 60F₂₅₄ made by Merck Co., developing solvent;chloroform:methanol:tetrahydrofuran=3:1:1)

FAB-MS(m/z):656(M+H)⁺

¹H-NMR(300 MHz,DMSO-d₆, δ ppm):11.18(1H,s),9.77(2H,br),8.84(1H,d,J=8.5Hz),8.76(1H,d,J=8.6 Hz),8.35(1H,d,J=1.7 Hz),7.72(2H,s),7.17(1H,d,J=1.7Hz),6.98(1H,d,J=1.9 Hz),6.78-6.98(2H,m),6.22(1H,t,J=4.6Hz),5.96(1H,d,J=8.9 Hz),5.87(1H,br),5.35(1H,br),5.22(1H,t,J=2.0Hz),5.11(1H,br),4.91(1H,br),4.46(2H,d,J=4.2 Hz),4.35(2H,d,J=4.6Hz),3.73-4.09(4H,m),3.49(2H, s)

Example 13

Compound represented by the structural formula

30 mg of Compound A and 30 mg of 3,4-bis-(t-butyldimethylsilyloxymethyl)thiophene-2-carbaldehyde were suspended in 6 ml of anhydrous methanol,30 ml of acetic acid was added, and the mixture was stirred at 80° C.for 2 hours. The reaction mixture was put on a Sephadex LH-20 column forchromatography and eluted with methanol. Fractions containing thedesired compound were concentrated to dryness to obtain 31 mg of anintermediate compound. This was suspended in 5 ml of methanol, 10 mg ofsodium cyanoborohydride and 100 ml of a 10% solution of hydrochloricacid in methanol were added, and the mixture was stirred at roomtemperature for 30 minutes. The reaction mixture was diluted with ethylacetate, and washed with water and an aqueous saturated sodium chloridesolution. The organic layer was dried and concentrated, and the residuewas put on a Sephadex LH-20 column for chromatography and eluted withmethanol. Fractions containing the desired compound were concentrated todryness to obtain 25 mg of the compound represented by the aboveformula.

Rf value: 0.30 (Kieselgel 60F₂₅₄ made by Merck Co., developing solvent;acetonitrile: tetrahydrofuran:toluene:water:acetic acid=4:2:2:0.5:0.1)

FAB-MS(m/z):691(M+H)⁺

¹H-NMR(300 MHz,DMSO-d₆, δppm):11.19(1H,s),9.79(1H,s),9.76(1H,s),8.86(1H,d,J=8.4Hz),8.78(1H,d,J=8.7 Hz),7.18(1H,d,J=1.8 Hz),7.15(1H,s),6.98(1H,d,J=2.1Hz),6.82(2H,dt,J=8.7,1.8 Hz),6.04(1H,t,J=5.4 Hz),5.97(1H,d,J=8.1Hz),5.86(1H,t,J=3.6 Hz),5.33(1H,d,J=4.2 Hz),5.12(1H,d,J=4.2Hz),5.01(1H,t,J=6.0 Hz),4.93(1H,d,J=4.8 Hz),4.85(1H,t,J=5.7Hz),4.52(2H,d,J=5.7 Hz),4.70(2H,d,J=5.7 Hz),4.40(2H,d,J=4.8Hz),4.01(1H,m),3.92(2H,m),3.77(1H,m),3.50(2H,m)

Example 14

Compound represented by the structural formula

18 mg of Compound A and 7 mg of 4-hydroxymethylpyridine-2-carbaldehydewere suspended in 2 ml of anhydrous methanol, several drops of aceticacid were added, and the mixture was stirred at 80° C. for 1.5 hours.The reaction mixture was concentrated to dryness, and the residue wasput on a Sephadex LH-20 column for chromatography and eluted withmethanol. Fractions containing the desired compound were concentrated todryness to obtain 22 mg of an intermediate compound. 90 mg of sodiumcyanoborohydride was suspended in 1 ml of tetrahydrofuran, and 0.66 mlof zinc chloride (1.0 M diethyl ether solution) was added dropwise. Asuspension of 22 mg of the intermediate compound in 3 ml oftetrahydrofuran was added, and the mixture was stirred at roomtemperature for 2.5 hours. Water was added to the reaction mixture, andthe mixture was made weakly alkaline with an aqueous saturated sodiumbicarbonate solution and extracted with ethyl acetate. The organic layerwas dried and concentrated, and the residue was put on a Sephadex LH-20column for chromatography and eluted with methanol. Fractions containingthe desired compound were concentrated to dryness to obtain 11 mg of thecompound represented by the above formula.

FAB-MS(m/z):656(M+H)⁺

¹H-NMR(300 MHz,DMSO-d₆, δppm):11.21(1H,s),9.80(1H,s),9.77(1H,s),8.86(1H,d,J=8.6Hz),8.78(1H,d,J=8.1Hz),7.82-7.95(1H,m),7.68-7.75(1H,m),7.33-7.43(1H,m),7.19(1H,s),7.00(1H,s),6.78-6.89(2H,m),6.22(1H,t,J=4.5Hz),5.97(1H,d,J=7.9 Hz),5.86(1H,t,J=3.8 Hz),5.33(1H,d,J=4.2Hz),5.29(1H,t,J=5.9 Hz),5.11(1H,d,J=5.0 Hz),4.91(1H,d,J=4.1Hz),4.42(2H,d,J=5.5 Hz),4.33(2H,d,J=1.6Hz),3.99-4.09(1H,m),3.91(2H,m),3.72-3.80(1H,m),3.50(2H,m)

Example 15

Compound represented by the structural formula

15 mg of Compound A and 6.9 mg of 6-hydroxymethylpyridine-2-carbaldehydewere suspended in 1 ml of anhydrous methanol, several drops of aceticacid were added, and the mixture was stirred at 80° C. for 5 hours. Thereaction mixture was concentrated to dryness, and the residue was put ona Sephadex LH-20 column for chromatography and eluted with methanol.Fractions containing the desired compound were concentrated to drynessto obtain 5.2 mg of an intermediate compound. 68 mg of sodiumcyanoborohydride was suspended in 2 ml of tetrahydrofuran, and 0.5 ml ofzinc chloride (1.0 M diethyl ether solution) was added dropwise. Asuspension of 5.2 mg of the intermediate compound in 1 ml oftetrahydrofuran was added, and the mixture was stirred overnight at roomtemperature. Water was added to the reaction mixture, and the mixturewas made weakly alkaline with an aqueous saturated sodium bicarbonatesolution and extracted with ethyl acetate. The organic layer was driedand concentrated, and the residue was put on a Sephadex LH-20 column forchromatography and eluted with methanol. Fractions containing thedesired compound were concentrated to dryness to obtain 2.0 mg of thecompound represented by the above formula.

Rf value: 0.29 (Kieselgel 60F₂₅₄ made by Merck Co., developing solvent:acetonitrile:tetrahydrofuran:toluene:water:acetic acid=4:2:2:0.5:0.1)

FAB-MS(m/z):656(M+H⁺)

¹H-NMR(300 MHz,DMSO-d₆, δppm):11.21(1H,s),9.80(1H,s),9.77(1H,s),8.86(1H,d,J=8.6Hz),8.78(1H,d,J=8.1Hz),7.82-7.95(1H,m),7.68-7.75(1H,m),7.33-7.43(1H,m),7.19(1H,s),7.00(1H,s),6.78-6.89(2H,m),6.22(1H,t,J=4.5Hz),5.97(1H,d,J=7.9 Hz),5.86(1H,t,J=3.8 Hz),5.33(1H,d,J=4.2Hz),5.29(1H,t,J=5.9 Hz),5.11(1H,d,J=5.0 Hz),4.91(1H,d,J=4.1Hz),4.42(2H,d,J=5.5 Hz),4.33(2H,d,J=1.6Hz),3.99-4.09(1H,m),3.91(2H,m),3.72-3.80(1H,m),3.50(2H,m)

Example 16

Compound represented by the structural formula

40 mg of Compound A and 60 mg of3,5-bis-(t-butyldimethylsilyloxymethyl)thiophene-2-carbaldehyde weresuspended in 8 ml of anhydrous methanol, 40 ml of acetic acid was added,and the mixture was stirred at 80° C. for 2 hours. The reaction mixturewas put on a Sephadex LH-20 column for chromatography and eluted withmethanol. Fractions containing the desired compound were concentrated todryness to obtain 46 mg of an intermediate compound. This was suspendedin 5 ml of methanol, 30 mg of sodium cyanoborohydride and 300 ml of a10% solution of hydrochloric acid in methanol were added, and themixture was stirred at room temperature for 30 minutes. The reactionmixture was diluted with ethyl acetate, and washed with water and anaqueous saturated sodium chloride solution. The organic layer was driedand concentrated, and the residue was put on a Sephadex LH-20 column forchromatography and eluted with methanol. Fractions containing thedesired compound were concentrated to dryness to obtain 36 mg of thecompound represented by the above formula.

Rf value: 0.24 (Kieselgel 60F₂₅₄ made by Merck Co., developing solvent:acetonitrile:tetrahydrofuran:toluene:water:acetic acid=4:2:2:0.5:0.1)

FAB-MS(m/z):690(M)⁺

¹H-NMR(300 MHz,DMSO-d₆, δppm):11.19(1H,s),9.79(1H,s),9.76(1H,s),8.86(1H,d,J=8.4Hz),8.78(1H,d,J=8.4 Hz),7.18(1H,d,J=1.8 Hz),6.98(1H,d,J=1.8Hz),6.83(1H,s),6.82(2H,dt,J=1.8,8.4 Hz),5.99(1H,t,J=4.8Hz),5.97(1H,d,J=9.0 Hz),5.87(1H,t,J=4.2 Hz),5.35(1H,d,J=5.2Hz),5.33(1H,t,J=4.8 Hz),5.12(1H,d,J=5.1 Hz),4.96(1H,d,J=5.7Hz),4.94(1H,d,J=5.4 Hz),4.49(4H,d,J=6.6 Hz),4.34(2H,d,J=4.8Hz),4.03(1H,m),3.92(2H,m),3.77(1H,m),3.50(2H,m)

Example 17

Compound represented by the structural formula

50 mg of Compound B and 60 mg of3,5-bis-(t-butyldimethylsilyloxymethyl)thiophene-2-carbaldehyde weredissolved in 12 ml of anhydrous methanol/N,N-dimethylformamide (5:1), 50ml of acetic acid was added, and the mixture was stirred overnight at80° C. The reaction mixture was diluted with ethyl acetate, washed withwater and an aqueous saturated sodium chloride solution, dried, andconcentrated. The residue was put on a Sephadex LH-20 column forchromatography and eluted with methanol. Fractions containing thedesired compound were concentrated to dryness, and the residue wasdissolved in 6 ml of a mixed solvent of tetrahydrofuran/methanol (2:1).30 mg of sodium cyanoborohydride and 300 ml of a 10% solution ofhydrochloric acid in methanol were added, and the mixture was at roomtemperature for 1 hour. The reaction mixture was diluted with ethylacetate, and washed with an aqueous saturated sodium bicarbonatesolution and an aqueous saturated sodium chloride solution. The organiclayer was dried and concentrated, and the residue was put on a SephadexLH-20 column for chromatography and eluted with methanol. Fractionscontaining the desired compound were concentrated to dryness to obtain37 mg of the compound represented by the above formula.

Rf value: 0.31 (Kieselgel 60F₂₅₄ made by Merck Co., developing solvent:acetonitrile:tetrahydrofuran:toluene:water:acetic acid=4:2:2:0.5:0.1)

FAB-MS(m/z):690(M)⁺

¹H-NMR(300 MHz,DMSO-d₆, δppm):10.90(1H,s),10.37(1H,s),9.98(1H,br),8.69(1H,d,J=8.4Hz),8.52(1H,d,J=8.4 Hz),7.19(1H,d,J=1.5,8.4 Hz),7.04(1H,d,J328.6Hz),7.01(2H,t,J=8.4 Hz),6.83(1H,s),6.04(1H,t,J=4.8Hz),5.42(1H,d,J=5.7 Hz),5.35(2H,d,J=6.0 Hz),5.21(1H,d,J=5.4Hz),4.95(1H,t,J=6.0 Hz),4.91(1H,br),4.50(4H,t,J=5.7Hz),4.36(2H,d,J=5.4Hz),4.00(2H,m),3.73(1H,m),3.62(2H,m),3.40(1H,m)

Example 18

Compound represented by the structural formula

20 mg of Compound A and 20 mg of 2-hydroxymethylthiophene-3-carbaldehydewere dissolved in 4 ml of anhydrous methanol, 20 ml of acetic acid wasadded, and the mixture was stirred at 80° C. for 2 hours. The reactionmixture was concentrated under reduced pressure, and the residue was puton a Sephadex LH-20 column for chromatography and eluted with methanol.Fractions containing the desired compound were concentrated to dryness,and the residue was dissolved in 3 ml of a mixed solvent oftetrahydrofuran/methanol (2:1). 30 mg of sodium cyanoborohydride and 300ml of a 10% solution of hydrochloric acid in methanol were added, andthe mixture was stirred at room temperature for 30 minutes. The reactionmixture was concentrated under reduced pressure, and the residue was puton a Sephadex LH-20 column for chromatography and eluted with methanol.Fractions containing the desired compound were concentrated to drynessto obtain 16 mg of the compound represented by the above formula.

Rf value: 0.49 (Kieselgel 60F₂₅₄ made by Merck Co., developing solvent:acetonitrile:tetrahydrofuran:toluene:water:acetic acid=4:2:2:0.5:0.1)

FAB-MS(m/z):660(M)⁺

¹H-NMR(300 MHz,DMSO-d₆, δppm):11.18(1H,s),9.79(1H,s),9.75(1H,s),8.86(1H,d,J=8.4Hz),8.78(1H,d,J=8.4 Hz),7.30(1H,d,J=4.8Hz),7.17(1H,d,J=2.1Hz),7.09(1H,t,J=5.4 Hz),6.98(1H,d,J=2.1Hz),6.82(1H,dd,J=2.1,8.4 Hz),6.80(1H,dd,J=2.1,8.4 Hz),5.97(1H,d,J=8.1Hz),5.92(1H,t,J=5.1 Hz),5.86(1H,t,J=3.9Hz),5.37(1H,d,J=5.7Hz),5.33(1H,d,J=4.5 Hz),5.12(1H,d,J=4.8Hz),4.93(1H,d,J=4.8 Hz),4.76(2H,d,J=5.7 Hz),4.20(2H,d,J=5.1Hz),4.00(1H,m),3.91(2H,s),3.77(1H,m),3.50(2H,m)

Example 19

Compound represented by the structural formula

30 mg of Compound A and 30 mg of3-t-butyldimethylsilyloxymethylthiophene-2-carbaldehyde were dissolvedin 5 ml of anhydrous methanol, 30 ml of acetic acid was added, and themixture was stirred 80° C. for 2 hours. The reaction mixture wasconcentrated under reduced pressure, and the residue was put on aSephadex LH-20 column for chromatography and eluted with methanol.Fractions containing the desired compound were concentrated to dryness,and the residue was suspended in 3 ml of methanol. 30 mg of sodiumcyanoborohydride and 300 ml of a 10% solution of hydrochloric acid inmethanol were added, and the mixture was stirred at room temperature for1 hours. The reaction mixture was put on a Sephadex LH-20 column forchromatography and eluted with methanol. Fractions containing thedesired compound were concentrated to dryness to obtain 24 mg of thecompound represented by the above formula.

Rf value: 0.40 (Kieselgel 60F₂₅₄ made by Merck Co., developing solvent:acetonitrile:tetrahydrofuran:toluene:water:acetic acid=4:2:2:0.5:0.1)

FAB-MS(m/z):660(M)⁺

¹H-NMR(300 MHz,DMSO-d₆, δppm):11.19(1H,s),9.78(1H,s),9.75(1H,s),8.85(1H,d,J=8.7Hz),8.78(1H,d,J=9.0 Hz),7.31(1H,d,J=4.8 Hz),7.18(1H,d,J=1.8Hz),699(1H,d,J=4.8 Hz),6.97(1H,d,J=1.8 Hz),6.81 (2H,dd,J=1.8,9.0Hz),6.06(1H,t,J=4.8 Hz),5.97(1H,d,J=8.7 Hz),5.86(1H,t,J=3.9Hz),5.33(1H,d,J=4.5 Hz),5.12(1H,d,J=4.5 Hz),4.99(1H,t,J=5.4Hz),4.93(1H,d,J=5.1 Hz),4.53(2H,d,J=5.7 Hz),4.38(2H,d,J=4.8Hz),4.02(1H,m),3.91(2H,m),3.77(1H,m),3.50(2H,m)

Example 20

Compound represented by the structural formula

30 mg of Compound A and 30 mg of4-t-butyldimethylsilyloxythiophene-3-carbaldehyde were suspended in 6 mlof methanol, 30 ml of acetic acid was added, and the mixture was stirred80° C. for 2 hours. The reaction mixture was put on a Sephadex LH-20column for chromatography and eluted with methanol. Fractions containingthe desired compound-were concentrated to dryness, and the residue wasdissolved in 5 ml of a mixed slovent of tetrahydrofuran/methanol (2:1).20 mg of sodium cyanoborohydride and 200 ml of a 10% solution ofhydrochloric acid in methanol were added, and the mixture was stirred atroom temperature for 30 minutes. The reaction mixture was diluted withethyl acetate, washed with water and an aqueous saturated sodiumchloride solution, dried and concentrated. The residue was put on aSephadex LH-20 column for chromatography and eluted with methanol.Fractions containing the desired compound were concentrated to drynessto obtain 15 mg of the compound represented by the above formula.

Rf value: 0.47 (Kieselgel 60F₂₅₄ made by Merck Co., developing solvent:acetonitrile:tetrahydrofuran:toluene:water:acetic acid=4:2:2:0.5:0.1)

FAB-MS(m/z):661 (M+H)⁺

¹H-NMR(300 MHz,DMSO-d₆, δppm):11.19(1H,s),9.79(1H,s),9.76(1H,s),8.86(1H,d,J=8.7Hz),8.78(1H,d,J=8.7 Hz),7.43(1H,d,J=3.3 Hz),7.30(1H,d,J=3.6Hz),7.17(1H,d,J=2.1 Hz),6.98(1H,d,J=2.1 Hz),6.83(1H,dd,J=2.1,8.7Hz),6.81(1H,dd,J=2.1,8.7 Hz),6.04(1H,t,J=5.1 Hz),5.97(1H,d,J=9.0Hz),5.87(1H,t,J=3.6 Hz),5.34(1H,d,J=3.9 Hz),5.12(1H,d,J=5.1Hz),5.10(1H,t,J=5.1 Hz),4.92(1H,d,J=4.5 Hz),4.67(2H,d,J=5.4Hz),4.23(2H,d,J=4.2 Hz),4.01(1H,m),3.92(2H,s),3.77(1H,m),3.50(2H,m)

Example 21

Compound represented by the structural formula

38 mg of Compound A and 25 mg of 4-hydroxymethylthiophene-2-carbaldehydewere suspended in 7 ml of anhydrous methanol, 45 ml of acetic acid wasadded, and the mixture was stirred at 80° C. for 7 hours. The reactionmixture was concentrated under reduced pressure, and 38 mg of crystalsobtained by filtration from methanol/chloroform. The crystals weredissolved in 10 ml of tetrahydrofuran/methanol (4:1), 13 mg of sodiumcyanoborohydride and 0.5 ml of a 10% solution of hydrochloric acid inmethanol were added, and the mixture was stirred at room temperature for30 minutes. The reaction mixture was diluted with ethyl acetate, washedwith an aqueous saturated sodium chloride solution, dried andconcentrated. The residue was put on a Sephadex LH-20 column forchromatography and eluted with methanol. Fractions containing thedesired compound were concentrated to dryness to obtain 21.7 mg of thecompound represented by the above formula.

Rf value: 0.24 (Kieselgel 60F₂₅₄ made by Merck Co., developing solvent:acetonitrile:tetrahydrofuran:toluene:water:acetic acid=4:2:2:0.5:0.1)

FAB-MS(m/z):660(M⁺)

¹H-NMR(300 MHz,DMSO-d₆, δ ppm):11.19(1H,s),9.77(2H,br),8.86(1H,d,J=8.6Hz),8.78(1H,d,J=8.6 Hz),7.17(1H,s),7.14(1H,d,J=1.8Hz),6.98(2H,m),6.81(1H,dt,J=18.,6.9 Hz),6.12(1H,t,J=5.1Hz),5.97(1H,d,J=8.1 Hz),5.87(1H,s),5.35(1H,d,J=1.8 Hz),5.13(1H,d,J=2.4Hz),5.01 (1H,t,J=5.4 Hz),4.93(1H,d,J=3.6 Hz),4.40(2H,d,J=4.5Hz),4.34(2H,d,J=4.8 Hz),4.00(1H,dd,J=2.1,11.6Hz),3.91(2H,s),3.79(1H,m),3.51 (2H,br)

Example 22

Compound represented by the structural formula

107 mg of Compound A and 126 mg of 5-hydroxymethylfurfural weresuspended in 2 ml of methanol, several drops of acetic acid were added,and the mixture was stirred overnight at 80° C. The reaction mixture wasconcentrated and the obtained solid was washed with chloroform. Thesolid was dissolved in 5 ml of a mixed solvent ofmethanol/tetrahydrofuran (1:2), 62.8 mg of sodium cyanoborohydride and 5ml of a 10% solution of hydrochloric acid in methanol were added, andthe mixture was stirred at room temperature for 30 minutes. The reactionmixture was diluted with a mixed slovent of ethyl acetate/methyl ethylketone, and washed with water and an aqueous saturated sodium chloridesolution. The organic layer was dried, concentrated, put on a SephadexLH-20 column for chromatography and eluted with methanol. Fractionscontaining the desired compound were concentrated to dryness to obtain103 mg of the compound represented by the above formula.

FAB-MS(m/z):644(M)⁺

¹H-NMR(300 MHz,DMSO-d₆, δ ppm):11.19(1H,s),9.77(2H,br),8.85(1H,d,J=8.5Hz),8.77(1H,d,J=8.6 Hz),7.18(1H,d,J=2.1 Hz),6.98(1H,d,J=1.7Hz),6.75-6.86(2H,m),6.31(1H,d,J=3.1 Hz),6.15(1H,d,J=3.1Hz),6.03(1H,t,J=4.7 Hz),5.97(1H,d,J=8.3 Hz),5.87(1H,t,J=3.6Hz),5.34(1H,d,J=3.9 Hz),5.08-5.15(2H,m),4.93(1H,d,J=4.5 Hz),4.28(2H,d,J=5.6 Hz),4.20(2H,d,J=4.7 Hz),3.72-4.05(4H,m),3.45-3.55(2H,m)

Example 23

Compound represented by the structural formula

40 mg of Compound A and 40 mg of 5-hydroxymethylthiophene-3-carbaldehydewere suspended in 8 ml of methanol, 40 ml of acetic acid was added, andthe mixture was stirred at 80° C. for 3 hours. The reaction mixture wascooled to room temperature, chloroform was added, and the resultingpowder was obtained by filtration. This was suspended in 5 ml ofmethanol, 20 mg of sodium cyanoborohydride and 200 ml of a 10% solutionof hydrochloric acid in methanol were added, and the mixture was stirredat room temperature for 30 minutes. The reaction mixture was dilutedwith a mixed slovent of ethyl acetate/methyl ethyl ketone, washed withwater and an aqueous saturated sodium chloride solution, dried, andconcentrated. The residue was put on a Sephadex LH-20 column forchromatography and eluted with methanol. Fractions containing thedesired compound were concentrated to dryness to obtain 21 mg of thecompound represented by the above formula.

Rf value: 0.29 (Kieselgel 60F₂₅₄ made by Merck Co., developing solvent:acetonitrile:tetrahydrofuran:toluene:water:acetic acid=4:2:2:0.5:0.1)

FAB-MS(m/z):660(M⁺)

¹H-NMR(300 MHz,DMSO-d₆, δ ppm):11.19(1H,s),9.80(2H,br),8.86(1H,d,J=9.0Hz),8.79(1H,d,J=8.7 Hz),7.34(1H,s),7.17(1H,d,J=1.5Hz),7.04(1H,s),6.97(1H,d,J=1.5 Hz),6.82(1H,dd,J=1.5,9.0Hz),6.80(1H,dd,J=8.7,1.5 Hz),5.97(2H,t,J=5.1Hz),5.87(1H,br),5.40(1H,t,J=6.0 Hz),5.35(1H,br),5.13(1H,s),4.91(1H,d,J=3.9 Hz),4.57(2H,d,J=4.2 Hz),4.20(2H,d,J=4.8Hz),3.88-4.10(3H,m),3.78(1H,m),3.50(2H,m)

Example 24

Compound represented by the structural formula

1.0 g of Compound A and 253 mg of 4-pyridinecarbaldehyde were dissolvedin 200 ml of methanol, 0.3 ml of acetic acid was added, and the mixturewas stirred overnight at 80° C. The deposited crystals were separated byfiltration, washed with chloroform, and dissolved in a mixed slovent ofmethanol/tetrahydrofuran (1:1). 10% palladium-carbon was added, and themixture was stirred overnight under a hydrogen stream. The mixture wasfiltered using Celite, and the filtrate was concentrated. The residuewas put on a Sephadex LH-20 column for chromatography and eluted withmethanol. Fractions containing the desired compound were concentrated todryness to obtain 730 mg of the compound represented by the aboveformula.

Rf value: 0.12 (Kieselgel 60F₂₅₄ made by Merck Co., developing solvent:toluene:acetonitrile:tetrahydrofuran:water:acetic acid=2:4:2:0.5:0.1)

FAB-MS(m/z):626(M+H)⁺

¹H-NMR(300 MHz,DMSO-d₆, δ ppm):11.18(1H,s),9.80(2H,br),8.89(1H,d,J=8.3Hz),8.76(1H,d,J=8.6 Hz),8.49(2H,dd,J=1.8,5.7 Hz),7.55(2H,d,J=6.0Hz),7.16(1H,d,J=1.5 Hz),6.97(1H,d,J=2.4 Hz),6.81(2H,dt,J=2.4,8.6Hz),6.32(1H,t,J=4.8 Hz),5.96(1H,d,J=9.0Hz),5.85(1H,br),5.34(1H,br),5.14(1H,br),4.90(1H,br),4.34(2H,d,J=4.2Hz),4.00(1H,d,J=11.1 Hz),3.90(2H,br),3.73˜3.80(1H,m),3.50(2H,br)

INDUSTRIAL APPLICABILITY

The compounds of the invention have an excellent antitumor effect, andare useful as antitumor agents in the pharmaceutical field.

What is claimed is:
 1. A compound represented by the formula:

wherein R represents an unsubstituted pyridyl, unsubstituted furyl orunsubstituted thienyl group, or a pyridyl, furyl or thienyl group eachof which has one or more substituents selected from the group consistingof a hydroxyl group, a lower alkoxy group, a hydroxy lower alkyl groupand a hydroxy lower alkenyl group except that when the pyridyl, furyl orthienyl group has a lower alkoxy group as a substituent, each of whichsimultaneously has another substituent selected from the groupconsisting of a hydroxyl group, a lower alkoxy group, a hydroxy loweralkyl group and a hydroxy lower alkenyl group, m represents an integerof 1 to 3, and G represents a β-D-glucopyranosyl group, and thepositions of substitution of the hydroxyl groups on theindolopyrrolocarbazole ring are the 1- and 11-positions, or the 2- and10-positions, or a pharmaceutically acceptable salt thereof.
 2. Thecompound according to claim 1 represented by the following formula orits pharmaceutically acceptable salt:


3. The compound according to claim 2 or its pharmaceutically acceptablesalt wherein R is a pyridyl group substituted with a hydroxy lower alkylgroup.
 4. The compound according to claim 3 or its pharmaceuticallyacceptable salt wherein R is a 6-hydroxymethylpyridin-2-yl group.
 5. Thecompound according to claim 3 or its pharmaceutically acceptable saltwherein R is a 5-hydroxymethylpyridin-4-yl group.
 6. The compoundaccording to claim 2 or its pharmaceutically acceptable salt wherein Ris a pyridyl group.
 7. The compound according to claim 6 or itspharmaceutically acceptable salt wherein R is a pyridin-4-yl group. 8.The compound according to claim 1 represented by the following formulaor its pharmaceutically acceptable salt:


9. A composition comprising a compound or its pharmaceuticallyacceptable salt according to claim 1, together with an excipient orcarrier.
 10. A method for treating cancer which comprises administeringto a mammal an effective amount of a compound or its pharmaceuticallyacceptable salt according to claim
 1. 11. The method according to claim10 wherein the mammal is a human.
 12. The method according to claim 11wherein the cancer treated is gastric cancer, colon cancer, lung canceror breast cancer.